Neuropathic pain in somatic and orofacial regions is difficult to treat, in part due to our incomplete understanding of cellular and molecular mechanisms underlying the genesis of neuropathic pain. Our work performed in the previous term of this grant has demonstrated how spinal cord microglial cells interact with neurons after nerve injury to drive neuropathic pain. We have demonstrated that activity-dependent activation of p38 MAPK in spinal cord microglia serves as a critical signaling for the genesis of neuropathic pain by producing the proinflammatory cytokines TNF-a and IL-1b. We have also demonstrated that TNF-a and IL-1b can powerfully regulate synaptic transmission in the spinal cord. However, mechanisms of neuronal-glial interactions after nerve injury are not fully understood. Recent studies show that caspases not only induce neuronal degeneration but also regulate synaptic plasticity. Our pilot studies have shown that (1) nerve injury up-regulates caspase-6 in spinal cord axonal terminals and (2) caspase-6 induces a substantial release of TNF-a in microglia. The overall goal of this competing renewal is to investigate how caspase-6 drives neuropathic pain via neuronal-glial interactions. We will test the following hypotheses via 4 specific aims using a mouse spinal nerve ligation model of neuropathic pain. Aim 1. Test the hypothesis that caspase-6 is both sufficient and required for the genesis of neuropathic pain and activation of spinal cord microglia; Aim 2. Test the hypothesis that nerve injury results in caspase-6 up-regulation in primary sensory neurons and spinal cord axons and induces caspase-6 release from primary sensory neurons; Aim 3. Test the hypothesis that caspase- 6 induces neuropathic pain by releasing TNF-a from microglia; and Aim 4. Test the hypothesis that caspase-6 rapidly enhances synaptic transmission and induces neuroplasticity in the spinal cord dorsal horn via TNF- a and microglial signaling. The proposed studies will use a multidisciplinary approach, including behavioral testing, siRNA knockdown, microglial culture, patch clamp recording in spinal cord slices and LTP recordings in intact animals, and caspase-6 and TNFR knockout mice to investigate caspase-6-induced neuronal-glial interactions after nerve injury. These studies will not only provide further insights into nerve injury-induced neuronal-glial interactions, but may also identify caspase-6 as a new target for treating neuropathic pain in the somatic and orofacial regions.